The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain

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Abstract
Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N.epsilon.-acetimidyl)19 cytochrome c, (N.epsilon.-isopropyl)19 cytochrome c, and (N.epsilon.,N.epsilon.-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N.epsilon.-isopropyl)19 and (N.epsilon.,N.epsilon.-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme. The cytochrome c derivatives with a single break in the polypeptide chain are partially present in a conformational state characterized by an opened heme cleft and a changed ligation state of the heme iron, the so-called ''''alkaline state'''' of cytochrome c. On binding to the oxidase, these derivatives are forced into a conformation which is very similar to that of the oxidase-bound native cytochrome c.

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